Specific lysis of target cells is a feature of a number of normal physiological processes. For example, CD8+ cytotoxic T lymphocytes (CTL) are central to the adaptive immune response against viruses, protozoa, intracellular bacteria, and in the rejection of allogeneic grafts. CTL recognize and kill target cells expressing antigen-derived peptides presented by class I major histocompatibility complex (MHC). In vivo, antigen specific CD8+ T cells also exert effector activity through release of soluble cytokines and chemokines, which either signal other effector cells or have direct antiviral activity. Other cells have lytic activity and participate in the elimination of infected cells or, in some circumstances, self derived cells as in autoimmune diseases. These lytic cells include CD4+ T lymphocytes with lytic effects, Natural Killer (NK) cells, Natural Killer T (NKT) cells and lymphokine activated killer (LAK) cells.
A number of cells that have cytotoxic potential express membrane receptors for the Fc region of an antibody molecule. When antibody is specifically bound to a target cell, receptor-bearing lytic cells bind to the target cell-bound antibody Fc region, and subsequently cause lysis of the target cell. Although the lytic cells are not themselves specific, the specificity of antibody directs them to the specific target cells, in a reaction known as antibody-dependent cell-mediated cytotoxicity (ADCC). Among the cells that can mediate ADCC are natural killer (NK) cell, macrophages, monocytes, neutrophils, and eosinophils. Antibodies can also be used to redirect effector lytic cells toward target cells.
Specific lysis of target cells is also a feature of certain aberrant physiological processes. Self-reactive T lymphocytes and antibodies are involved in the destruction of cells in autoimmune disorders such as insulin-dependent (Type 1) diabetes mellitus and autoimmune hemolytic anemia. Graft-versus-host disease (GVDH) develops when immunocompetent lymphocytes are injected into an allogeneic recipient whose immune system is compromised, e.g., a patient who has had radiation exposure or who has leukemia, immunodeficiency disease, or an autoimmune anemia and who is a recipient of an allogeneic bone marrow transplant. The grafted lymphocytes attack the host, whose immunocompromised state prevents an adequate immune response against the graft.
The cytolytic activity of CD8+ T cells has been commonly determined by the 51Chromium (51Cr) release assay. The standard 51Cr release assay has a number of disadvantages that include high spontaneous release, influence of 51Cr upon the effector cell population, problems with labeling certain cell types, low sensitivity, and health risks associated with gamma irradiation. Assays such as the modified enzyme linked immuno-assay (ELISPOT), which detects the secretion of cytokines following antigenic stimulation, and the use of tetrameric MHC class I complexes have afforded greater sensitivity in the detection of antigen specific CD8+ T cells.
Non-radioactive alternatives to the standard cytotoxic assays have included detection of released intracellular enzymes, colorimetric assays or detailed preparation of reporter cell lines. In addition, assays that employ the use of flow cytometry by the detection of fluorescent dyes for either lymphocyte-target conjugate formation or cytolytic activity have been described. Flow cytometric cytotoxicity assays generally involve the measurement of a fluorochrome released from, or remaining in, pre-labeled effector cells or targets, simultaneously or exclusively. Ideally, these labels should not change the morphology or function of the labeled cells. However, some of the fluorochromes that have been proposed label target cells poorly, require complex manipulations of light scatter properties to discriminate the viable cell population or have a higher spontaneous release compared to standard 51Cr release. In addition, fluorometric techniques which rely on the incorporation of a nucleic acid stain such as propidium iodide, do not account for active phagocytic cells that can take up dead cells in vitro. As a consequence, none of these proposed assays have gained acceptance, or replaced the 51 Cr release assay, which remains in widespread use.
To improve the study of in vitro cytolytic function of lytic agents such as cytolytic cells and antibodies, there is a need for a reliable assay that analyzes subpopulations, eliminates the requirement for potentially hazardous radioactive isotopes, offers increased sensitivity, utilizes an efficient label that is detected easily with low spontaneous release and is reproducible. The present invention addresses this need.
LITERATURE
Lebow et al. (1986) Nat. Immun. Cell Growth Regul. 5:221–237; Cavarec et al. (1990) J. Immunol. Methods 130:251–261; Lebow and Bonavida (1990) Proc. Natl. Acad. Sci. USA 87:6063–6067; Callewaert et al. (1991) Cytometry 12:666–676; Radcliff et al. (1991) J. Immunol. Methods 139:281–292; Vitale et al. (1992) J. Immunol. Methods 149:189–196; Radosevic et al. (1993) J. Immunol. Methods 135:81–89; VanGraft et al. (1993) Cytometry 14:257–264; King and Radicchi-Mastroianni (1996) Cytometry 24: 368–373; Zamai et al. (1998) Cytometry 32:280–285; Shau and Dawson (1985) J. Immunol. 135:137–140; Larsson and Nygren (1989) Anticancer Res. 9: 1111–1119; Chang et al. (1993) J. Immunol. Methods 166:45–54; Hatam et al. (1994) Cytometry 16: 59–68; Karawajew et al. (1994) J. Immunol. Methods 177:119–130; Papa and Valentini (1994) Methods Cell Bio. 42: 193–207; Papadopoulos et al. (1994) J. Immunol. Methods 177:101–111; Flieger et al. (1995) J. Immunol Methods 180:1–13; Johann et al. (1995) J. Immunol Methods 185:209–216; Mattis et al. (1997) J. Immunol. Methods 204:135–142; Chang et al. (1998) J. Immunol. Methods 211:51–63; Aubry et al. (1999) Cytometry 37:197–204; Goldberg et al. (1999) J. Immul Methods 224:1–9; Nociari et al. (1998) J. Immunol. Methods 213:157–167; Slezak and Horan (1989) J. Immunol. Methods 117:205–214; Radosevic et al. (1990) J. Immul. Methods 135:81–89; Mattis et al. (1997) J. Immunol. Methods 204:135–142; Schafer et al. (1997) J Immul. Methods 204:89–84; Flieger et al. (1999) Hybridoma 18:63–68; Beavis and Pennline (1994) J. Immunol. Methods 170:57–65; Ford et al. (1996) J. Surg. Res. 62:23–28; Garton and Schoenwolf (1996) Anatomical Record 244:112–117; Hasbold et al. (1998) Immunol. Cell Biol. 77:516–522; Parish (1999) Immunol Cell Biol. 77:499–508; Song et al. (1999) Transplantation 68:297–299; Renno et al. (1999) J. Immunol. 162:6312–6315.